Modeling Charcot-Marie-Tooth Disease <em>In Vitro</em> by Transfecting Mouse Primary Motoneurons

Arnaud Jacquier; Val&#233;rie Risson; Laurent Schaeffer

doi:10.3791/57988

Modelado enfermedad del Charcot-Marie-diente In Vitro por transferencia primaria Motoneu...

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Neuroscience

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JoVE Journal Neuroscience

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

Modelado enfermedad del Charcot-Marie-diente In Vitro por transferencia primaria Motoneurons de ratón

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07:43 min

January 7, 2019

DOI: 10.3791/57988-v

Arnaud Jacquier^1,2, Valérie Risson¹, Laurent Schaeffer^1,3

¹Institut NeuroMyoGène, CNRS UMR5310, INSERM U1217,Université Lyon, ²Unité Fonctionnelle de Neurogénétique Moléculaire,Hospices Civils de Lyon, ³Centre de Biotechnologie Cellulaire,Hospices Civils de Lyon

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Overview

This study presents a method for isolating and transfecting primary motoneurons (MNs) from murine spinal cords. It focuses on understanding motor neuron polarity and neurodegenerative mechanisms associated with motoneuron diseases through enriched culture techniques.

Key Study Components

Area of Science

Neuroscience
Cell Biology
Neurodegenerative Diseases

Background

Motor neurons are essential for muscle control and are affected in various neurodegenerative diseases.
Understanding the biology of motoneurons aids in addressing diseases like amyotrophic lateral sclerosis.
The enrichment of motoneuron cultures allows for targeted studies of cellular mechanisms.
Transfection techniques enable the study of gene functions and disease modeling.

Purpose of Study

To develop a reliable method for obtaining a highly enriched culture of motoneurons.
To facilitate the study of pathological mechanisms implicated in motoneuron disorders.
To investigate the effects of genetic mutations using transfection methodologies.

Methods Used

Cell culture techniques were applied to isolated murine spinal cord fragments.
Primary motoneurons were enriched through a density gradient centrifugation method.
The protocol includes enzymatic digestion and several centrifugation steps for cellular purification.
Magnetofection was employed for transfecting neurons with DNA constructs.
Imaging techniques were used to analyze cell morphology post-transfection.

Main Results

The described method successfully enriched motoneuron populations for further analysis.
Transfected motoneurons exhibited distinct morphological characteristics and protein expression indicative of their identities.
Findings highlight potential pathways involved in motoneuron diseases and illustrate cellular responses to genetic modifications.

Conclusions

This study provides a robust framework for investigating motoneuron biology and disease mechanisms.
The techniques developed offer significant insights into cellular processes relevant to motoneuron disorders.
Overall, this method enhances our understanding of neurodegenerative processes and facilitates future therapeutic explorations.

Frequently Asked Questions

What are the advantages of this method for studying motoneurons?

The technique allows for the rapid enrichment and isolation of motoneurons, enabling focused studies on their biology and disease mechanisms while minimizing contamination from other cell types.

How are primary motoneurons isolated from the spinal cord?

Motoneurons are isolated through a multi-step process involving dissection, enzymatic digestion, and density gradient centrifugation, ensuring a high purity of the target cell population.

What types of outcomes can be assessed using this technique?

This method allows for assessments of cellular morphology, protein expression, and the effects of genetic modifications on motoneuron function and survival.

How can this methodology be adapted for other types of neurons?

With modifications to the dissection and culture protocols, this approach can be applied to isolate other neuronal subtypes for similar investigations.

What considerations should be kept in mind while using this method?

Researchers should be mindful of the timing and conditions during enzymatic digestion, as well as the centrifugation speeds, to optimize cell yield and viability.

What insights can this study provide for neurodegenerative research?

The enriched cultures can reveal critical insights into the cellular mechanisms underlying various motoneuron diseases and facilitate the testing of potential therapeutic approaches.

El objetivo de esta técnica es preparar una cultura altamente enriquecida de primarias motoneuronas (MNs) de murine de la médula espinal. Para evaluar las consecuencias de las mutaciones que causan enfermedades de MN, Describimos aquí el aislamiento de estos aislados MNs y su transfección por magnetofection.

Este método puede ayudar a abordar cuestiones clave en neurobiología, como la polaridad de las neuronas motoras, la orientación de axón, el tráfico de axones y los mecanismos neurodegenerativos. La principal ventaja de esta técnica es obtener un cultivo de neuronas motoras altamente enriquecido mientras tomamos para expresar nuestra proteína knockdown por minuto de afecto. Para comenzar este procedimiento, coloque un embrión en un plato de medios de disección codificados con silicio.

Bajo el microscopio, sostenga el embrión con el abdomen contra el silicio con fórceps. Con el segundo par de fórceps, inserte una de las puntas en el canal central de la médula espinal rostrad. Luego, cierre los fórceps para desgarrar ligeramente el tejido dorsal.

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